ONCOLOGY LETTERS 13: 1932-1938, 2017

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MicroRNA-134 targets KRAS to suppress breast cancer cell proliferation, migration and invasion XIAOMEI SU*, LING ZHANG*, HUA LI, PENG CHENG, YAJIE ZHU, ZHEN LIU, YU ZHAO, HONGYU XU, DONG LI, HUI GAO and TAO ZHANG Department of Oncology, Chengdu Military General Hospital, Chengdu, Sichuan 610083, P.R. China Received July 19, 2015; Accepted September 12, 2016 DOI: 10.3892/ol.2017.5644 Abstract. The expression patterns and functions of microRNA‑134 (miR‑134) have been previously studied in numerous types of cancer. To the best of our knowledge, this is the first study of miR‑134 in human breast cancer. In the present study, the expression patterns, biological functions and underlying molecular mechanisms of miR‑134 in human breast cancer were investigated. Reverse transcription‑quantitative polymerase chain reaction evaluated the expression of miR‑134 in human breast cancer tissues, matched normal adjacent tissues, breast cancer cell lines and a normal mammary epithelial cell line. Following transfection with miR‑134, an MTT assay, cell migration assay, cell invasion assay, western blot analysis and a luciferase assay were performed on the MCF‑7 and MDA‑MB‑231 human breast cancer cell lines. The findings revealed that miR‑134 expression levels were significantly downregulated in breast cancer cells. Statistical analysis demonstrated that low expression of miR‑134 was significantly associated with lymph node metastasis, TNM stage and reduced cell differentiation. It was observed that miR‑134 inhibited the growth, migration and invasion of breast cancer cells. Additionally, the present study indicated that miR‑134 may directly target the Kirsten rat sarcoma viral oncogene homolog in breast cancer tissues. These results suggest that miR‑134 may be used as a potential therapeutic biomarker in breast cancers. Introduction Breast cancer is the most common malignancy among women and the leading cause of cancer‑associated mortality,

Correspondence to: Professor Tao Zhang, Department of Oncology, Chengdu Military General Hospital, 270 Rongdu Road, Chengdu, Sichuan 610083, P.R. China E‑mail: [email protected] *

Contributed equally

Key words: microRNA‑134, breast cancer, Kirsten rat sarcoma viral oncogene homolog, therapy

accounting for 14% of global cases (1,2). In the United States, there were ~234,190 recorded new cases and 40,730 mortalities due to breast cancer in 2015 (3). The etiology of breast cancer remains unclear, although genetic and epigenetic alterations are considered to contribute to tumorigenesis and progression (4). Despite advances in modern therapies for patients with breast cancer, including surgery, radiotherapy, hormonal therapy and various types of chemotherapeutic approaches using targeted and non‑targeted drugs, numerous patients with breast cancer respond only transiently to conventional chemotherapy (5,6). A high proportion of these patients eventually exhibit tumor metastasis, which is a major contributor to cancer mortality (5,6). Therefore, it is necessary to explore the molecular mechanisms underlying the tumorigenesis and progression of breast cancer in order to develop more effective treatments. Numerous previous studies have demonstrated that microRNAs (miRNAs) are involved in the tumorigenesis and progression of breast cancers (7‑9). miRNAs are a novel group of non‑protein‑coding, single‑stranded, short (generally 18‑24 nucleotides in length) proteins, which regulate the translational or post‑transcriptional levels of target mRNAs, through binding to the 3'‑untranslated region (UTR) of those target mRNAs  (10). These miRNAs are located in the introns of non‑coding genes, the introns of protein‑coding genes or the exons of non‑coding genes (11). miRNAs have crucial functions in various physiological and pathological processes, including cell proliferation, survival, migration, invasion and the cell cycle (12). Previous studies have suggested that specific miRNAs may be downregulated or upregulated in certain types of tumors (13‑15). Downregulated miRNAs may normally function as tumor suppressor genes, whereas upregulated miRNAs may normally function as oncogenes (16). Dysregulated miRNA expression has been observed in various types of human malignancies, including breast cancers (17). These previous studies indicated that specific dysregulated miRNAs may serve as useful biomarkers for breast cancer tumorigenesis, progression and clinical prognosis, as well as potential targets for breast cancer therapy (16,17). The expression patterns and underlying mechanisms of miR‑134 have been previously studied in various types of cancer. However, to the best of our knowledge, this is the first study of miR‑134 in human breast cancer (18‑20).

SU et al: miR-134 IN BREAST CANCER

In our current study, we examined miR‑134 expression in breast cancer tissues and cell lines. The association between miR‑134 expression and clinicopathological features was also analyzed. In addition, the effects of miR‑134 on breast cancer cell proliferation, migration and invasion were evaluated. Furthermore, the molecular mechanism underlying the biological roles of miR‑134 on breast cancer cells was explored. The results of the current study have potential therapeutic applications and may be used to develop current and novel treatments for breast cancers. Materials and methods Ethics statement and clinical specimens. The Ethics Committee of the Chengdu Military General Hospital approved the present study. At initial diagnosis, written informed consent was obtained from all patients. A total of 85 pairs of breast cancer tissue and normal adjacent tissue (NAT) samples were obtained from patients (age range, 23‑82 years) who had undergone breast surgery at the Chengdu Military General Hospital (Chengdu, China). In the current study, the patients involved had not received chemotherapy or radiotherapy prior to breast surgery. Clinicopathological data for these patients, including age, tumor diameter, lymph node metastasis, TNM stage and pathological differentiation, were also collected. The tissue samples were snap frozen in liquid nitrogen immediately following surgery and stored at ‑80˚C until use in the present study. Cell culture and transfection. The MCF‑7 and MDA‑MB‑231 breast cancer cell lines, and the MCF‑10A normal mammary epithelial cell line, were acquired from the American Type Culture Collection (Manassas, VA, USA). All cell lines were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or RPMI‑1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37˚C in an atmosphere containing 5% CO2 and 100% humidity. miR‑134 mimics, negative control (NC) and luciferase reporter plasmids were synthesized by GenePharma Co. Ltd. (Shanghai, China). Cells were seeded in a six‑well plate at 40‑50% confluence. Following overnight incubation, the cells were transfected with miR‑134 mimics or NC using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 nmol/l. RNA isolation and reverse transcription‑quantitative poly‑ merase chain reaction (RT‑qPCR). Total RNA was isolated from the tissue samples and cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentration was determined with NanoDrop ND‑1000 Spectrophotometer. Equal amounts of RNA were subjected to cDNA synthesis using a PrimeScript RT Regent kit (Takara, Bio, Inc., Otsu, Japan). RT‑qPCR was subsequently performed using a SYBR green kit (Takara Bio, Inc.) with U6 as an internal control, according to the manufacturer's protocol. The reaction system contained 10 µl SYBR Green I mix, 2 µl cDNA, 2 µl forward primer, 2 µl reverse primer and 4 µl ddH 2O. The thermal cycling

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conditions of the reaction were as follows: 95˚C for 10 min; and 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. U6 RNA was used as an internal control. Primers were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). All RT‑qPCR was performed in ABI 7500 RT‑qPCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). All assays were performed in triplicate. MTT assay. An MTT assay was used to investigate the effect of miR‑134 on breast cancer cell growth. Transfected cells (miR‑134 and NC) were seeded into 96‑well plates at a density of 3x103 cells/well. The cells were incubated with 20 µl MTT (5 mg/ml; Sigma‑Aldrich, St. Louis, MO, USA). Following incubation for 4 h at 37˚C, the formazan precipitates were dissolved in 200 µl dimethyl sulfoxide. The absorbance at 490 nm was evaluated using an ELISA reader (Bio‑Rad Laboratories, Inc., Hercules, CA, USA). All experiments were repeated in triplicate. Cell migration and invasion assay. To investigate the effect of miR‑134 on cell migration and invasion, Transwell chambers with an 8‑µm pore polycarbonate membrane (Costar; Corning Incorporated, Corning, NY, USA) were used. Diluted Matrigel (50 µl; 2 mg/ml; BD Biosciences, San Jose, CA, USA) was placed on the inner chamber membrane surface for the invasion assay. Transfected cells were collected, counted and resuspended in single cell suspension (FBS‑free culture medium). Subsequently, 1x105 cells were added to the upper chamber and 500 µl culture medium supplemented with 20% FBS, was added into the lower chamber as a chemoattractant. Following a 24 h incubation, any non‑migrated cells were carefully removed from the top of the chamber using a cotton swab. Subsequently, the chambers were fixed with 100% methanol, stained with 0.5% crystal violet (Beyotime Institute of Biotechnology, Haimen, China) for 10 min and washed with PBS (Gibco; Thermo Fisher Scientific, Inc.) 3 times. The chambers were photographed and counted in five random fields under a light microscope with x200 magnification using Photoshop (Adobe, San Jose, CA, USA). All experiments were repeated in triplicate. miR‑134 target prediction. The target genes of miR‑134 were predicted by using TargetScan (version 7.0; http://www. targetscan.org/index. html) (21). Western blotting. A western blot analysis was used to quantify the changes in Kirsten rat sarcoma viral oncogene homolog (KRAS) protein expression levels. Cells transfected with miR‑134 and NC were lysed with a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined by using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc., Rockford, IL, USA). An equal amount of protein (20  µg) from each cell line was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and the proteins were subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non‑fat dry milk (Beyotime Institute of Biotechnology) in Tris‑buffered saline. The membranes were then incubated with a mouse anti‑human

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ONCOLOGY LETTERS 13: 1932-1938, 2017

monoclonal KRAS primary antibody at a 1:500 dilution (cat. no. ab157255; Abcam, Cambridge, MA, USA) overnight at 4˚C. After washing with Tris‑buffered saline with 0.5% Tween 20 (Beyotime Institute of Biotechnology) 3 times, the membranes were incubated with a goat anti‑mouse horseradish peroxidase‑conjugated secondary antibody (1:1,000 dilution; cat. no. ab97023; Abcam) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence solution (Pierce Biotechnology, Inc., Rockford, IL, USA). Glyceraldehyde 3‑phosphate dehydrogenase was used as an internal control. The protein blots were quantified using AlphaEase FC software (Cell Biosciences, Inc., San Jose, CA, USA).

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Luciferase assay. To determine whether KRAS was a direct target of miR‑134, a luciferase assay was used. Cells were seeded in 12‑well plates at 30‑40% confluence, and then were transfected with miR‑134 mimics or NC, following which the cells were co‑transfected with a reporter plasmid containing the wild‑type (Wt) and mutant 3'‑UTR of KRAS; Lipofectamine ® 2000 was used as a transfection reagent. Following a 48 h transfection, a luciferase assay was performed using a dual‑luciferase reporter assay system (Promega Corporation, Madison, WI, USA). The firefly luciferase activity was normalized to the corresponding Renilla luciferase activity. All Luciferase assays were repeated in 3 independent experiments.

B

Statistical analysis. Data were presented as the mean ± standard deviation. Data were compared with SPSS 17 software (SPSS, Inc., Chicago, IL, USA) using a Student's t‑test. A 2‑tailed value of P

MicroRNA-134 targets KRAS to suppress breast cancer cell proliferation, migration and invasion.

The expression patterns and functions of microRNA-134 (miR-134) have been previously studied in numerous types of cancer. To the best of our knowledge...
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